1. Field of the Invention
The present invention relates to genes which are differentially expressed during banana fruit development, the protein products of these genes, and DNA regulatory elements which are differentially expressed during banana fruit development.
2. Description of the Related Art
Bananas represent a crop of great importance to both the world economy and as a means of supplying subsistence to a large portion of the world's population. The global banana export market is about 10% of the world's production with a $4 billion dollar value. Banana fruit are the fourth most important food in the developing world (May, G D et al. (1995) Biotechnology 13:486-492) with approximately 100 million people acquiring their main energy source from bananas. Bananas, like kiwifruit, papayas, and apples, are climacteric fruit, meaning they ripen in association with an ethylene signal. In the ripening process, starch degradation is associated with a respiratory climacteric in the fruit. Banana fruit ripening is characterized by a number of biochemical and physiological changes including fruit softening, changes in peel color and an increase in respiratory activity (Seymour, G B (1993) in: Seymour G B, et al. (eds) Biochemistry of Fruit Ripening, pp 83-106. Chapman & Hall, London). Although ethylene is produced by the fruit, ripening can also be stimulated by the application of exogenous ethylene. Alternatively, endogenous ethylene production may be stimulated, e.g., by exposing fruit to acetylene.
More specifically, the post-harvest physiology of the banana (Musa acuminata cv. Grand Nain) is characterized by initial harvest, a green storage phase, followed by a burst in ethylene production that signals the beginning of the climacteric period. Associated with this respiratory climacteric is a massive conversion of starch to sugars in the pulp, during which the activities of enzymes involved in starch biosynthesis decrease while those involved in starch breakdown and mobilization increase rapidly (Wu et al. (1989) Acta Phytophysiol. Sin. 15:145-152; Agravante et al. (1990) J. Jpn. Soc. Food Sci. Technol. 37:911-915; Iyare et al. (1992) J. Sci. Food Agric. 58: 173-176; Cordenunsi et al. (1995), J. Agric. Food Chem. 43:347-351; Hill et al. (1995) Planta 196:335-343 and 197:313-323). In addition, the rate of respiration rises sharply (Beaudry et al. (1987) Plant Physiol. 8:277-282; Beaudry et al. (1989) Plant Physiol. 91:1436-1444).
Other changes that occur during ripening include: fruit softening as a result of enzymatic degradation of structural carbohydrates (Agravante et al. (1991) J. Jpn. Soc. Food Sci. Technol. 38:527-532; Kojima et al. (1994) Physiol. Plant. 90:772-778); a decline in those polyphenol compounds responsible for the astringency of the green unripe fruit which are catalyzed by polyphenol oxidase and peroxidases (Mendoza et al. (1994) in I Uritani et al., eds., Postharvest Biochemistry of Plant Food-Materials in the Tropics. Japan Scientific Societies Press, Tokyo, pp 177-191); an increase in the activity of alcohol acetyltransferase, the enzyme that catalyzes the synthesis of isoamyl acetate--the major aroma compound of banana fruit (Harada et al. (1985) Plant Cell Physiol. 26:1067-1074); and a de-greening of the peel as a result of chlorophyll breakdown by chlorophyllase (Thomas et al. (1992) Int. J. Food Sci. Technol. 27:57-63). Stages of banana fruit ripening are scored by peel color index (PCI) numbers, on a scale from 1--very green, to 7--yellow-flecked with brown flecks (Color Preferences Chart, Customer Services Department, Chiquita Brands, Inc.,). PCI can be correlated with other biochemical and physiological parameters associated with fruit development and ripening such as ethylene biosynthesis and respiratory rate. The respiratory rate usually peaks at PCI 2 and PCI 4, respectively, in ethylene-treated bananas (Agravante et al. (1991) supra).
Associated with the respiratory climacteric is a large increase in the rate of protein synthesis (Mugugaiyan (1993) Geobios, 20:18-21), as well as differential protein accumulation (Dominguez-Puigjaner et al. (1992) Plant Physiol. 98:157-162). Poly-galacturonase (PG) has been identified as a protein that increases in banana pulp during ripening, as determined by 2-D gel electrophoresis and immuno-hybridization (id.). Many of the changes that occur during ripening require de novo protein synthesis (Areas et al. (1988) J. Food Biochem. 12:51-60); therefore, a secondary approach to investigate changes that occur during a ripening is to isolate transcripts encoding proteins associated with the ripening process. Analogous studies of differential gene expression have been successfully employed in other plant species.
Other enzymes associated with developing and ripening of fruit include proteinase inhibitors and chitinases (Dopico et al. (1993) Plant Molec. Bio. 21:437), stress-related enzymes (Ledger et al. (1994) Plant Molec. Biol. 25:877), .beta.-oxidation pathway enzymes (Bojorquez et al. (1995), Plant Molec. Biol. 28:811), and metabolite-detoxifying enzymes (Picton et al. (1993) Plant Molec. Biol. 23:193). Chitinases are abundant proteins found in a wide variety of plants. Although chitinases are produced by a diversity of plant species, the presence of chitin has not been reported in higher plants. Since chitin is the major structural component of fungal cell walls, it has been proposed that chitinases serve as defense proteins with antifungal activity. Chitinases are reported to be induced in higher plants by a number of different types of stress (Linthorst (1991) Crit. Rev. Plant Sci. 10:123; Punja et al. (1993) J. Nematol. 25:526; Collinge et al. (1993) Plant J. 3:31). Many plant chitinases are expressed constitutively, although at a low level.
As noted above, in ripening climacteric fruit, starch degradation is associated with a respiratory climacteric in the fruit. Reactive oxygen species (ROS) are byproducts of cellular respiration, especially under conditions which result in high levels of NADH. ROS generation during respiration may be at the site of ubiquinones in the electron transport chain. Both yeast and mammalian metallothioniens may play a direct role in the cellular defense against oxidative stress by functioning as antioxidants (Dalton et al. (1994) Nucl. Acids Res. 22:5016-5203; Tamai et al. (1993) Proc Nat Acad Sci (USA) 90:8013-8017; Bauman et al. (1991) Toxicol. Appl. Pharmacol. 110:347-354). MT may play an additional role in supplying metal ions to Cu- and Zn-superoxide dismutase (SOD), an enzyme that catalyzes the disproportionation of superoxide anion to hydrogen peroxide and dioxygen and is thought to play an important role in protecting cells from oxygen toxicity.
Transcripts encoding MT or MT-like proteins have been isolated from many different plants (recently reviewed in Robinson et al. (1993) Biochem J. 295:1-10). There is accumulating evidence that the plant MT mRNAs are translated, and the protein may have a function in the plant tissues from which transcripts have been isolated. A seed-associated polypeptide (E.sub.c protein) has been purified from wheat and sequenced (Kawashima et al. 1992), and more recently, MT was reported to have been isolated from Arabidopsis (meeting abstract). Based on deduced amino acid sequences, plant MT proteins are approximately 70 aa and have characteristic cysteine-rich regions at the N and C termini, separated by a variable spacer region. Based on the number and distribution of the cysteine residues, plant MTs have been classified into two distinct types (Robinson et al. (1993), supra). Type 1 MTs have 6 N-terminal and 6 C-terminal cysteine residues, whereas type 2 have 8 cysteine residues in the N-terminal domain and 6 at the C-terminus. Although there are no strict patterns of MT expression, in general type 1 transcript abundance is high in roots, and is often metal-inducible, whereas type 2 is expressed primarily in leaves. Other transcripts have been isolated that encode proteins with homology to plant MTs but cannot be classified as either type 1 or type 2, and these include seed-specific proteins or transcripts from barley and wheat (see, Robinson et al. (1993), supra). In Arabidopsis thaliana, MT proteins are encoded by a gene family containing five members, two copies encoding a type 2 MT and 3 encoding a MT with homology to type 1 (Zhou et al. (1995) Mol. Gen. Genet. 248:318-328).
In plants transcripts encoding metallothionein-like proteins have often been isolated by differential screening. Type 2 MT have recently been isolated from plants expressed in association with senescence, leaf abcission (Coupe et al. (1995) Planta 197:442447), and fruit ripening (Ledger et al. (1994) Plant Molec. Biol. 25:877-886). Using differential screening, Ledger and Gardner (id.) found transcripts encoding MT-like proteins in developing kiwifruit. One, pKIWI503, was specifically upregulated late in fruit development, during ripening of the mature fruit.
A major component of the export market is the level of ripening control which is exerted by modern banana shipping systems. Bananas for export must be shipped under refrigeration at 12-14.degree. C., often under controlled atmosphere (CA) conditions (i.e., low oxygen combined with CO.sub.2) which reduces the effects of ethylene produced by the fruit. Exposure to ethylene for 24 hours at concentrations of 100-1000 .mu.l per liter is used to trigger the ripening climacteric. This "gassing" step is typically done near the final point in the distribution system. Although this system is entirely functional, resulting in marketability of high quality fruit with minimal losses, there remains a role for engineered ethylene control in the banana export market. Bananas for export are harvested green at approximately 75% of full size. This is done to ensure, even with the use of low temperature and CA, that few if any of the bananas start ripening during shipment. Allowing the bananas to remain on the plant longer would result in more carbohydrate accumulation to the fruit and a direct, zero cost increase in yield. If engineered ethylene control were implemented in banana, this increased yield would come at no increased risk of premature ripening during shipment.
Moreover, linking exogenous genes to isolated gene promoters that are differentially expressed during banana ripening, and in response to ethylene, would allow for the production of exogenous protein in banana tied to the ripening process, and in other plants, controlled by ripening or exposure to ethylene.